ABSTRACT Surgical site infections (SSIs) within 30 days of an operation contribute substantially to surgical morbidity and mortality each year. The majority of these infections involve antibiotic-resistant ESKAPE pathogens. These bacteria thrive in healthcare settings and traditional antibiotics cannot kill them as they are either multi-drug or extremely drug resistant. The increasing frequency of these pathogens underscores the priority for developing novel approaches to supplement the current antimicrobial regimens used in the prevention of surgical site and wound infections. The Centers for Medicare and Medicaid Services no longer reimburses healthcare facilities for care related to SSIs. Any intervention that reduces the threat of SSIs could save thousands of lives and billions of dollars in healthcare costs. To address this research priority, we outline a strategy to develop a therapeutic anti-infective wound dressing with a novel mode of action that could greatly reduce rates of infections in wounds and surgical sites and possibly decrease development of antibiotic resistance. Recently, we developed unique vitamin D3-loaded poly(?-caprolactone) (PCL) nanofibers via electrospinning and showed release of vitamin D for more than 28 days in vitro. This induced higher levels of antimicrobial peptide LL-37 expression in monocytes and skin cells than free vitamin D3 and enhanced killing of bacteria by monocytes. In this application, we seek to develop technologies that prevent infections associated with SSIs or traumatic injuries by local sustained co- delivery of vitamin D3 and other immune-boosting compounds. Our strategy to achieve this goal is four-fold: 1) Establish effective encapsulation of vitamin D3 with additional immune-boosting compounds in nanofiber wound dressings of different sizes, structures and drug loadings and examine their release profiles. Also, establish effective gas-foaming expansion of nanofiber dressings in the third dimension; 2) Optimize drug loadings and release profiles from nanofiber dressings for vitamin D in combination with other immune-boosting compounds and determine efficacy for inducing bacterial-killing activity of skin and immune cells in cell culture; 3) Test the efficacy of these nanofiber-based wound dressings to i) protect ?humanized? transgenic mice with skin wounds from bacterial infection by drug-resistant S. aureus and ii) promote wound healing; and 4) Test the efficacy of these nanofiber-based wound dressings to protect human skin wounds from bacterial infection ex vivo. We expect to lay the foundation for developing an inexpensive intervention with great potential that could reduce infections, speed healing and greatly improve quality of care, decrease costs and most importantly save the lives of patients. We expect that boosting the immune system to attack pathogens from multiple directions will provide a strategy that is effective against drug-resistant pathogens and less likely to select for drug-resistance.